Gloria Peiró

Gut Microbiota Dysbiosis Is Associated with Inflammation and Bacterial Translocation in Mice with CCl4-Induced Fibrosis

Background Gut is the major source of endogenous bacteria causing infections in advanced cirrhosis. Intestinal barrier dysfunction has been described in cirrhosis and account for an increased bacterial translocation rate. Hypothesis and Aims We hypothesize that microbiota composition may be affected and change along with the induction of experimental cirrhosis, affecting the inflammatory response. Animals and Methods Progressive liver damage was induced in Balb/c mice by weight-controlled oral administration of carbon tetrachloride. Laparotomies were performed at weeks 6, 10, 13 and 16 in a subgroup of treated mice (n = 6/week) and control animals (n = 4/week). Liver tissue specimens, mesenteric lymph nodes, intestinal content and blood were collected at laparotomies. Fibrosis grade, pro-fibrogenic genes expression, gut bacterial composition, bacterial translocation, hosts specific butyrate-receptor GPR-43 and serum cytokine levels were measured. Results Expression of pro-fibrogenic markers was significantly increased compared with control animals and correlated with the accumulated dose of carbon tetrachloride. Bacterial translocation episodes were less frequent in control mice than in treated animals. Gram-positive anaerobic Clostridia spp count was decreased in treated mice compared with control animals and with other gut common bacterial species, altering the aerobic/anaerobic ratio. This fact was associated with a decreased gene expression of GPR43 in neutrophils of treated mice and inversely correlated with TNF-alpha and IL-6 up-regulation in serum of treated mice along the study protocol. This pro-inflammatory scenario favoured blood bacterial translocation in treated animals, showing the highest bacterial translocation rate and aerobic/anaerobic ratio at the same weeks. Conclusions Gut microbiota alterations are associated with the development of an inflammatory environment, fibrosis progression and bacterial translocation in carbon tetrachloride-treated mice.

Cellular apoptosis susceptibility gene expression in endometrial carcinoma: correlation with Bcl-2, Bax, and caspase-3 expression and outcome.

Deregulation of proliferation and apoptosis is known to contribute to neoplastic transformation and growth. Using specific antibodies for the cellular apoptosis susceptibility (CAS) gene, caspase-3, Bcl-2, and Bax, we examined the protein expression in 89 endometrial carcinomas and in 56 samples of nonneoplastic adjacent endometrium for comparison. Immunostaining results were scored with regard to approximate percentage of positive tumor cells (<10%, 10% to 50%, >50%) and relative immunostaining intensity (1+, 2+, 3+). In nonneoplastic endometrium, CAS protein was expressed in 70.6%, Bax in 64%, caspase-3 in 52%, and Bcl-2 in 87%. In neoplastic tissue, CAS was present in 93% of the tumors, Bax in 88%, caspase-3 in 77%, and Bcl-2 in 51%. Bcl-2:Bax ratio was >1 in 9 cases (10%). In cases of atrophy (n=24) and simple (n=10) and complex (n=22) hyperplasia in the adjacent endometrium, lower levels of expression compared with carcinoma were observed for CAS (p=0.003), caspase-3 (p=0.034), and Bax (p=0.04) and higher levels for Bcl-2, although for this protein the results were not statistically significant (p=0.32). There was no association between immunoscores and FIGO stage. High caspase-3 levels were seen in endometrioid tumor type (p=0.017). CAS expression was higher in grade 3 tumors (p=0.002) and older patients (p=0.013). All tumors of younger patients (<50 years) were Bcl-2 negative (p=0.037). Caspase-3 correlated positively with CAS (p=0.008), Bax (p=0.04), and low Bcl-2:Bax ratio (p=0.043), and inversely (as a trend) with Bcl-2 (p=0.056). Survival analysis (Kaplan-Meier and Cox regression) established a strong association between prognosis and stage, grade, and histologic type (all p≤0.0036). In addition, shorter survival was observed for patients whose tumors contained >50% of positive cells for caspase-3 (p=0.024) or for CAS (p=0.04). Age, Bcl-2, Bax, and Bcl-2:Bax ratio did not provide prognostic information. Our results suggest a role of CAS, Bcl-2, Bax, and caspase-3, which are apparently involved in the progressive deregulation of proliferation and apoptosis leading from simple and complex hyperplasia to carcinoma. In addition, CAS and caspase-3 protein levels may be useful markers in predicting the outcome of the patients.

20q13 and cyclin D1 in ovarian carcinomas. Analysis by fluorescence in situ hybridization

In ovarian carcinomas, alterations of the chromosomal region 20q13 and the cyclin D1 gene have been described. This study has sought to determine their prognostic significance. Fluorescence in situ hybridization (FISH) on dissociated nuclei and paraffin sections with DNA probes for 20q13.2 and cyclin D1, as well as immunohistochemistry (cyclin D1), were applied to formalin‐fixed tissue of 69 invasive ovarian carcinomas, mainly of serous type. On dissociated nuclei 33/47 cases (70%) and on tissue sections 13/66 cases (20%) demonstrated an increase of 20q13.2 copies. The presence of ≥4 copies per nucleus (isolated nuclei) and ≥3 copies per nucleus (sections) was associated with an adverse prognosis (Kaplan–Meier for FIGO stage III after stratification for residual tumour: p=0.0049 and p=0.03, respectively). Thirty‐four out of 47 cases (72%) showed an increase of cyclin D1 copies. Kaplan–Meier analysis for FIGO stage III after stratification for residual tumour>2 cm or ≤2 cm revealed an unfavourable outcome for cases with more than two cyclin D1 copies (p=0.04). No correlation was seen between FISH and immunohistochemistry. Multivariate analysis identified residual tumour (p=0.0002), 20q13.2 gain (p=0.0004) and cyclin D1 gain (p=0.0343) as independent prognostic factors. It is concluded that gains of chromosomal region 20q13.2 and the cyclin D1 gene are frequent and biologically important events, with prognostic relevance, in advanced ovarian carcinomas. Copyright

Analysis of HER-2/neu amplification in endometrial carcinoma by chromogenic in situ hybridization. Correlation with fluorescence in situ hybridization, HER-2/neu, p53 and Ki-67 protein expression, and outcome.

Fluorescence in situ hybridization (FISH) is the most widely used technique to detect HER-2/neu gene amplification; however, it is only available in some institutions. In contrast, chromogenic in situ hybridization (CISH) can be evaluated by routine light microscopy. In endometrial carcinoma there are few data concerning HER-2/neu status and prognosis. Therefore, we determined HER-2/neu gene status by CISH using a digoxigenin-labelled probe on 60 formalin-fixed paraffin-embedded endometrial carcinomas. The data were compared with the immunohistochemistry of HER-2/neu (A0485, TAB250), p53, Ki-67, clinicopathological factors, and survival. By conventional light microscopy, HER-2/neu amplification (⩾6 copies >50% cancer cells) was detected in 14% (8/59) tumours, HER-2/neu overexpression (>10% cells moderate/strong complete membrane staining) in 22% (13/60) for A0485, and 18% (11/60) for TAB250, p53 (>10% +cells) in 61% (36/59), and Ki-67 (>50% +cells) in 50% (30/60). Discordant cases for CISH and immunohistochemistry, as well as all (2+) were further analysed by FISH (Vysis). Among 10 cases (2+) and not amplified by CISH, two showed low-level amplification by FISH. Significant correlation was found between amplification and protein overexpression (P⩽0.001), and a trend with nonendometrioid type, higher grade, and older age. A better outcome (Kaplan–Meier) was observed for patients with nonamplified (1–5 copies per nucleus) or low-level (6–10 copies) amplification tumours, low Ki-67 expression, age <50 years, endometrioid type, low FIGO (International Federation of Obstetrics and Gynaecology) grade and stage, superficial myometrial infiltration, and no lymph–vascular invasion (P⩽0.036), but only as a trend for HER-2/neu protein negative (P=0.13). Cox analysis revealed age, FIGO grade and stage, myometrial infiltration, and lymph–vascular invasion to be independent prognostic factors (P⩽0.05), and a trend for HER-2/neu gene copy number (0.18). In endometrial carcinoma, HER-2/neu gene status can be readily assessed by CISH in routine clinical practice, and it gives more prognostic information than HER-2/neu by immunohistochemistry. FISH analysis in (2+) cases but negative by CISH may detect additional tumours with low-level amplification.

CAS (Cellular Apoptosis Susceptibility) Gene Expression in Ovarian Carcinoma Correlation With 20q13.2 Copy Number and Cyclin D1, p53, and Rb Protein Expression

We immunohistochemically analyzed cellular apoptosis susceptibility (CAS) protein expression and compared it with 20q13.2 copy number and the expression of cell cycle-associated proteins retinoblastoma (Rb), cyclin D1, and p53 and prognosis on paraffin-embedded tissue from 69 ovarian carcinomas (OCs). CAS protein reactivity was present in 100%, Rb in 54%, cyclin D1 in 47%, and p53 in 49%. Significant reciprocal correlation was observed between high levels of CAS and histologic type, FIGO (International Federation of Obstetrics and Gynecology) stage III and grade 3, residual tumor (>2 cm), 20q13.2 (ZNF217 gene) amplification (>4 copies in >20% cells), and high expression of cyclin D1 (all P < .05). No association was found between cyclin D1, p53, or Rb levels with clinicopathologic factors. In univariate analysis, residual tumor, FIGO stage and grade, ZNF217 amplification, and CAS levels predicted outcome (all P < .05). In multivariate analysis, stage, grade, amount of residual tumor, and ZNF217 amplification showed independent prognostic value (all P < .05). In OC, alteration of CAS and ZNF217 genes, both located at 20q13, is frequent and relevant prognostically. Cyclin D1, Rb, and p53 seem to have a secondary role.

Insulin-like growth factor-I receptor and PTEN protein expression in endometrial carcinoma. Correlation with bax and bcl-2 expression, microsatellite instability status, and outcome.

We immunohistochemically analyzed 89 endometrial carcinomas for insulin-like growth factor-I receptor (IGF-IR) and PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression. Results were compared with clinicopathologic factors, bcl-2 and bax expression, microsatellite instability (MSI) status, and prognosis. Increased expression of IGF-IR and bcl-2 (> 50% cells) was seen in 60 cases (67%) and 15 cases (17%), respectively; loss of PTEN was seen in 13 cases (15%) and of bax in 11 cases (12%). No significant correlation was observed between the proteins or with clinicopathologic factors. Loss of PTEN was more frequent in MSI-positive tumors (4/10 [40%]) than in negative tumors (9/79 [11%]; P = .016). Longer survival was observed for patients with endometrioid tumors, International Federation of Gynecology and Obstetrics stage I or II tumors, grade 1 tumors, superficial myometrial infiltration (< or = 50%), less than 5% necrosis, no vascular invasion, or low level IGF-IR (< 10% of cells) (P < or = .05). Cox analysis showed independent value only for stage, grade, type, and lymph-vascular invasion (P < .05). Our data demonstrate that IGF-IR overexpression occurs in a subset of endometrioid carcinomas, which has potential prognostic value, while loss of PTEN often is associated with the MSI phenotype.

Insulin-like Growth Factor-I Receptor and PTEN Protein Expression in Endometrial Carcinoma

We immunohistochemically analyzed 89 endometrial carcinomas for insulin-like growth factor-I receptor (IGF-IR) and PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression. Results were compared with clinicopathologic factors, bcl-2 and bax expression, microsatellite instability (MSI) status, and prognosis. Increased expression of IGF-IR and bcl-2 (>50% cells) was seen in 60 cases (67%) and 15 cases (17%), respectively; loss of PTEN was seen in 13 cases (15%) and of bax in 11 cases (12%). No significant correlation was observed between the proteins or with clinicopathologic factors. Loss of PTEN was more frequent in MSI-positive tumors (4/10 [40%]) than in negative tumors (9/79 [11%]; P = .016). Longer survival was observed for patients with endometrioid tumors, International Federation of Gynecology and Obstetrics stage I or II tumors, grade 1 tumors, superficial myometrial infiltration (≤50%), less than 5% necrosis, no vascular invasion, or low level IGF-IR (<10% of cells) (P ≤ .05). Cox analysis showed independent value only for stage, grade, type, and lymph-vascular invasion (P < .05). Our data demonstrate that IGF-IR overexpression occurs in a subset of endometrioid carcinomas, which has potential prognostic value, while loss of PTEN often is associated with the MSI phenotype.

Prognostic Relevance of hMLH1, hMSH2, and BAX Protein Expression in Endometrial Carcinoma

Endometrial carcinoma is the most common gynecologic malignancy in perimenopausal and postmenopausal women. A role of mismatch repair genes, like hMLH1 and hMSH2 in their pathogenesis, has been suggested. Loss of their function leads to the accumulation of replication errors (mutator phenotype), which are responsible for further mutations in genes with microsatellite sequences in their coding region, such as Bax. We analyzed the expression of hMLH1, hMSH2, and Bax genes in 89 formalin-fixed paraffin-embedded endometrial carcinomas. The immunostains were scored with regard to percentage of positive tumor cells (0%, <10%, 10 to 50%, >50%), and relative staining intensity (1+, 2+, 3+). The staining results were correlated with clinicopathologic features and survival. Loss of hMSH2 expression (0% positive cells) was observed in 1.1% (1/89) of the tumors; loss of hMLH1 was seen in 12.4% (11/89) of the cases, particularly in endometrioid tumors with mucinous differentation (5/11; 45%; P = .03). No significant association was found between the immunoscores and grade, stage criteria of the International Federation of Obstetrics and Gynecology (FIGO), or age of the patients. Among 11 tumors with loss of Bax expression (12.4%), 4 had also loss of hMLH1 (4/11; 36.4%; P = .017). In multivariate analysis (Cox model), significantly longer survival was found for patients with tumors in FIGO Stage I–II (P < .0001), endometrioid type (P = .001), low grade (P = .001), and absence of hMLH1 expression (P = .027). Our results suggest that loss of function of hMLH1 and Bax occur in a subgroup of endometrial carcinoma. In addition to the classical prognostic factors, absence of hMLH1 expression is associated with better outcome of patients.

Prognostic Implications of HER-2 Status in Steroid Receptor–Positive, Lymph Node–Negative Breast Carcinoma

Patients with lymph node-negative breast carcinoma (LNNBC) and positive hormone receptor (HR) status during estrogen-based endocrine therapy have different prognoses. The contribution of HER-2 (human epidermal growth factor receptor-2) has not been extensively evaluated. We stained 230 LNNBCs for estrogen and progesterone receptors (ER and PR) and HER-2. HER-2 gene status was studied in 150 randomly selected tumors by chromogenic in situ hybridization and cases with discordant or nondefinitive results by fluorescence in situ hybridization. Patients with ER+ and/or PR+ tumors were treated with tamoxifen. We found positive expression of ER, PR, and HER-2 in 73.7%, 67%, and 27.8%, respectively, and HER-2 amplification in 18.0%. Poorer outcome was seen for patients with ER+ and/or PR+/HER-2 overexpressing tumors and as a trend for patients with HER-2 amplification. ER/PR and HER-2 expression showed an independent prognostic value. In LNNBCs with positive HR status, HER-2 overexpression and/or amplification confer an aggressive tumor phenotype, and this might be related to tamoxifen unresponsiveness.

Beta-adrenergic receptor 1 selective antagonism inhibits norepinephrine-mediated TNF-alpha downregulation in experimental liver cirrhosis.

Background Bacterial translocation is a frequent event in cirrhosis leading to an increased inflammatory response. Splanchnic adrenergic system hyperactivation has been related with increased bacterial translocation. We aim at evaluating the interacting mechanism between hepatic norepinephrine and inflammation during liver damage in the presence of bacterial-DNA. Animals and Methods Forty-six mice were included in a 16-week protocol of CCl4-induced cirrhosis. Laparotomies were performed at weeks 6, 10, 13 and 16. A second set of forty mice injected with a single intraperitoneal dose of CCl4 was treated with saline, 6-hydroxidopamine, Nebivolol or Butoxamine. After 5 days, mice received E. coli-DNA intraperitoneally. Laparotomies were performed 24 hours later. Liver bacterial-DNA, norepinephrine, TNF-alpha, IL-6 and beta-adrenergic receptor levels were measured. Results Bacterial-DNA translocation was more frequent in CCl4-treated animals compared with controls, and increased as fibrosis progressed. Liver norepinephrine and pro-inflammatory cytokines were significantly higher in mice with vs without bacterial-DNA (319.7±120.6 vs 120.7±68.6 pg/g for norepinephrine, 38.4±6.1 vs 29.7±4.2 pg/g for TNF-alpha, 41.8±7.4 vs 28.7±4.3 pg/g for IL-6). Only beta-adrenergic receptor-1 was significantly increased in treated vs control animals (34.6±7.3 vs 12.5±5.3, p = 0.01) and correlated with TNF-alpha, IL-6 and norepinephrine hepatic levels in animals with bacterial-DNA. In the second set of mice, cytokine levels were increased in 6-hydroxidopamine and Nebivolol (beta-adrenergic receptor-1 antagonist) treated mice compared with saline. Butoxamine (beta-adrenergic receptor-2 antagonist) didn’t inhibit liver norepinephrine modulation of pro-inflammatory cytokines. Conclusions Beta-adrenergic receptor-1 mediates liver norepinephrine modulation of the pro-inflammatory response in CCl4-treated mice with bacterial-DNA.